In silico screening and evaluation of the anticonvulsant activity of docosahexaenoic acid-like molecules in experimental models of seizures

Background: Resistance to antiepileptic drugs and the intolerability in 20-30% of the patients raises demand for developing new drugs with improved efficacy and safety. Acceptable anticonvulsant activity, good tolerability, and inexpensiveness of docosahexaenoic acid [DHA] make it as a good candidate for designing and development of the new anticonvulsant medications

Methods: Ten DHA-based molecules were screened based on in silico screening of DHA-like molecules by root-mean-square deviation of atomic positions, the biological activity score of Professional Association for SQL Server, and structural requirements suggested by pharmacophore design. Anticonvulsant activity was tested against clonic seizures induced by pentylenetetrazole [PTZ, 60 mg/kg, i.p.] and tonic seizures induced by maximal electroshock [MES, 50 mA, 50 Hz, 1 ms duration] by intracerebroventricular [i.c.v.] injection of the screened compounds to mice

Results: Among screened compounds, 4-Phenylbutyric acid, 4-Biphenylacetic acid, phenylacetic acid, and 2-Phenylbutyric acid showed significant protective activity in pentylenetetrazole test with ED[50] values of 4, 5, 78, and 70 mM, respectively. In MES test, shikimic acid and 4-tert-Butylcyclo-hexanecarboxylic acid showed significant activity with ED[50] values 29 and 637 mM, respectively. Effective compounds had no mortality in mice up to the maximum i.c.v. injectable dose of 1 mM

Conclusion: Common electrochemical features and three-dimensional spatial structures of the effective compounds suggest the involvement of the anticonvulsant mechanisms similar to the parent compound DHA

Hippocampal expression of connexin36 and connexin43 during epileptogenesis in pilocarpine model of epilepsy

Background: Gap junctions [GJs] provide direct intercellular communications that are formed by hexameric protein subunits, called connexin [Cx]. The role of Cxs in epileptogenesis has not received sufficient attention. Hippocampus with critical function in epileptogenesis has a wide network of GJs. We examined the protein expression levels of hippocampal Cx36 [the prominent Cx present between GABAergic interneurons] and Cx43 [the main Cx expressed by astrocytes] during epileptogenesis in the pilocarpine model of epilepsy

Methods: Male Wistar rats received scopolamine [1 mg/kg, s.c.]. Pilocarpine [380 mg/kg, i.p.] was administered 30 min thereafter to induce status epilepticus [SE]. SE was stopped 2 h later by diazepam [10 mg/kg, i.p.]. Cx36 and Cx43 protein expression was assessed by Western blot analysis in the hippocampus of SE-experienced rats, after injection of diazepam [F0 subgroup], after acquisition of focal seizures [F3 subgroup], and after development of generalized seizures [F5 subgroup]. The control subgroups, C0, C3, and C5, were aged-matched rats, which received saline [1 ml/kg, i.p.] instead of pilocarpine. Injection of scopolamine and diazepam, and dissection of hippocampi were carried out at the same time interval as the test subgroups

Results: SE emerged in 67.1% of pilocarpine-treated animals. Focal and generalized seizures developed 3.8 +/- 0.4 and 7.0 +/- 0.5 days after SE, respectively. Cx36 protein abundance was not significantly different between test and control groups in the three time points. However, Cx43 protein level showed 40% increase in F3 subgroup [P<0.05 compared to C3, P<0.01 compared to F0 and F5]

Conclusion: Hippocampal Cx43 is overexpressed in pilocarpine model of epileptogenesis after acquisition of focal seizures

Facilitation of hippocampal kindling and exacerbation of kindled seizures by intra-CA1 injection of quinine: a possible role of Cx36 gap junctions

Background: GABAergic interneurons in the hippocampal CA1 area are mutually communicated by gap junctions [GJs] composed of connexin36 [Cx36]. We examined the role of Cx36 in CA1 in manifestation of kindled seizures and hippocampal kindling in rats

Methods: Quinine, as the specific blocker of Cx36, was injected into CA1, and kindled seizures severity was examined 10 min afterward. Moreover, quinine was injected into CA1 once daily, and the rate of CA1 kindling was recorded

Results: Quinine 0.5 and 1 mM caused 2- and 3.5-fold increase in the duration of total seizure behavior and generalized the seizures. Primary and secondary after discharges [AD] were also significantly increased. Quinine 0.1 mM augmented the rate of kindling and the growth of secondary AD

Conclusion: Cx36 GJs in CA1 are the main components of hippocampal inhibitory circuit. Any interruption in this path by pathologic or physical damages can trigger hippocampal hyperexcitability and facilitate epileptogenesis

Anticonvulsant effect of cicer arietinum seed in animal models of epilepsy: introduction of an active molecule with novel chemical structure

Cicer arietinum [Chickpea] is one of the most important harvests in the world with high nutritional value. Lack of essential oils in the seeds of Chickpea is an advantage in search for drug-like molecules with less toxicity. We evaluated anticonvulsant effect of C. arietinum in common animal models of epilepsy. Dichloromethane extract was obtained from C. arietinum seeds by percolation. Acute toxicity of the extract was assessed in mice. Protective effect of the extract was examined against tonic seizures induced by maximal electroshock [MES; 50 mA, 50 Hz, 1 s] in mice, clonic seizures induced by pentylenetetrazole [PTZ; 60 mg/kg, i.p.] in mice, and electrical kindling model of complex partial seizures in rats. The extract was fractionated by nhexane to f1 and f2 fractions. The extract and fractions underwent phytochemical analysis by thin layer chromatography. The active anticonvulsant fraction, f1, was subjected to matrix assisted laser desorption/ionization [MALDI] mass analysis. The crude extract had neither toxicity up to 7 g/kg nor protective activity in MES and kindling models. However, it significantly inhibited clonic seizures induced by PTZ. f1 fraction mimicked protective effect of the extract. Phytochemical screening revealed the presence of considerable amount of alkaloids in the extract and fractions. Moreover, a novel structural class was detected in f1 fraction. Finding an anticonvulsant molecule pertaining to a new structural class in the seeds of C. arietinum promises an effective and inexpensive source of antiepileptic medication. Further studies are needed to identify its mechanism of action and more clues into its structure-activity relationship.

Effect of chronic intracerebroventricluar administration of lipopolysaccharide on connexin43 protein expressionin rat hippocampus

Hippocampal damages, which are accompanied by inflammation, are among the main causes of epilepsy acquisition. We previously reported that chronic intracerebroventricular [i.c.v.] injection of lipopolysaccharide [LPS] modulates epileptogenesis in rats. There is a network of gap junction channels in the hippocampus that contribute to epileptogenesis. Gap junction channels are formed by oligomeric protein subunits called connexins [Cx]. Astrocytic Cx43 and neuronal Cx36 are expressed in the hippocampus. In order to find out the possible role of gap junctions in seizure-modulating effect of LPS and neuroinflammation, we studied the effect of central administration of LPS on expression of Cx36 and Cx43 in rat hippocampus. LPS, 2.5 micro g/rat/day, was injected i.c.v. to male Wistar rats for 14 days. mRNA and protein abundance of Cx36, Cx43 and IL1-beta were measured in rat hippocampus by real time-PCR, Western blot and ELISA techniques, at the beginning, in the middle, and at the end of the treatment period. IL1-beta protein level was significantly increased 6 h after first injection of LPS. Cx36 and Cx43 mRNA expression did not alter during chronic administration of LPS. A selective decrease in Cx43 protein expression was observed after 7 injections of LPS. It is suggested that Cx43 containing gap junctions in the hippocampus is down-regulated in response to chronic injection of LPS. This event can inhibit propagation of toxic and noxious molecules to neighboring cells and modulate hippocampal excitability and epileptogenesis

Upregulation of connexins 30 and 32 gap junctions in rat hippocampus at transcription level by chronic central injection of lipopolysaccharide

Gap junctions composed of connexins [Cx] are functional in cell defense by propagation of toxic/death molecules to neighboring cells. Hippocampus, one of the brain regions with particular vulnerability to damage, has a wide network of gap junctions. Functional response of astrocytic Cx30 and neuronal Cx32 to hippocampal damage is unknown. We infused lipopolysaccharide [LPS] intracerebroventricularly [2.5 microg/rat] once daily for two weeks to create neuroinflammation. The mRNA and protein levels of the Cx were measured in the hippocampus after 1[st], 7[th] and 14[th] injection by real-time PCR and Western-blot techniques. A significant increase in Cx32 and Cx30 gene expression was observed after 7[th] and 14[th] injection of LPS with no significant change in their protein abundance. Transcriptional overexpression of hippocampal Cx30 and Cx32 could be an adaptive response to production of intracellular toxic molecules but it is not accompanied with post- transcriptional overexpression and might have no functional impact

Effect of lntra-amygdala injection of lipopolysaccharide on kindling epileptogenesis in adult rats

Contribution of neuroinflammation and epilepsy in the mature brain has elicited contradictory results with either excitatory or inhibitory effects. The amygdala is one of the main parts of the limbic system susceptible to insults that lead to neuroinflammation and epilepsy. This study evaluates the effect of chronic inflammation of the rat amygdala induced by lipopolysaccharide [LPS] on kindling epileptogenesis. LPS [5microg/rat] was infused once daily into the basolateral amygdala [BLA] of adult rats. Daily electrical stimulation [150-300 microA, 100 Hz, monophasic square wave stimulus of 1 msec per wave, 2 sec duration] was delivered into BLA 30 min after LPS injections until the animals became fully kindled. LPS had no significant effect on the development of focal and generalized seizures. The type of neural system exposed to LPS and its specific electrophysiological properties seems to ascertain the final excitatory or inhibitory outcome

Association between ABCB1-T1236C polymorphism and drug-resistant epilepsy in Iranian female patients

One third of epileptic patients are resistant to several anti-epileptic drugs [AED]. P-glycoprotein [P-gp] is an efflux transporter encoded by ATP-binding cassette subfamily B member 1 [ABCB1] gene that excludes drugs from the cells and plays a significant role in AEDs resistance. Over-expression of P-gp could be a result of polymorphisms in ABCB1 gene. We studied the association of T129C and T1236C single-nucleotide polymorphisms [SNP] of ABCB1 gene with drug-resistant epilepsy in Iranian epileptics. DNA samples were obtained from 200 healthy controls and 332 epileptic patients, of whom 200 were drug responsive and 132 drug resistant. The frequencies of the genotypes of the two SNP were determined by polymerase chain reaction followed by restriction fragment length polymorphism. No significant association was found between T129C and T1236C genotypes and drug-resistant epilepsy neither in adults nor in children. However, the risk of drug resistance was higher in female patients with 1236CC [P = 0.02] or CT [P = 0.008] genotype than in those with TT genotype. The risk of drug resistance was also higher in patients with symptomatic epilepsies with 1236CC [P = 0.02] or CT [P = 0.004] genotype than in those with TT genotype. The risk of drug resistance was lower in patients with idiopathic epilepsies with 129TT genotype [P = 0.001] than in those with CT genotype. Our results indicate that T1236C polymorphism is associated with drug resistance in Iranian female epileptic patients. Replication studies with large sample sizes are needed to confirm our results

Antinociceptive and anti-inflammatory activity of the seed and root extracts of ferula gummosa boiss in mice and rats

Ferula gummosa Boiss [Apiaceae] has been used in Iranian traditional medicine for the relief of stomach pain. In this study, effects of aqueous, methanolic and acetone extracts of the seed and root of this plant in experimental models of acute pain [Tail-flick=TF], chronic pain [Formalin test=FT] and inflammation [Cotton pellet granuloma=CPG] was investigated. The results showed that the highest non-sedative dose of each of these three extracts had no effect in TF. Among the extracts, only the acetone extract of the root could reduce licking and biting time in the late phase of FT, although this effect might be to some extent due to the solvent [Tween 80]. None of the extracts had anti-inflammatory effect in CPG. Preliminary phytochemical analysis of methanol and acetone extracts showed presence of terpenoids and alkaloids and small amounts of cardenolids. The results of our study suggest further evaluation of antinociceptive and anti-inflammatory effects of other kinds of extracts in order to determine the best extract with highest efficacy and lowest side effects

Anticonvulsant effect of ferula gummosa root extract against experimental seizures

In Iranian traditional medicine, there are some report regarding the anticonvulsant effect of Ferula gummosa Boiss. In this study, anticonvulsant activity, neurological deficits and lethality of the root acetone extract of this plant were evaluated in mice. The extract exhibited dose-dependent prevention of tonic seizures induced by pentylenetetrazole [ED50 = 154.4 mg/kg]. However, the extract produced sedation and motor impairment with TD50 value of 546 mg/kg. Preliminary phytochemical analysis showed the presence of terpenoids, alkaloids and a little amount of cardenolids in the extract. It seems that the anticonvulsant and neurotoxic effects of the extract is related in part to the terpenoid compounds. The acceptable protective index of the extract [3.5] recommends further studies on Ferula gummosa

Anti - nociceptive effect of the fruit essential oil of cuminum cyminum L. in rat

Cuminum cyminum Linn. [Umbelliferae] is a plant, which has been used as a toothache remedy in folk medicine of Iran. In this study, the potential anti-nociceptive and anti-inflammatory activities of the fruit essential oil of C. cyminum has been evaluated in chemical [formalin test] and thermal [tail-flick test] models of nociception and formalin model of acute inflammation in rats and mice. The essential oil at the doses ranging between 0.0125 and 0.20 ml/kg exhibited a significant and dose-dependent analgesic effect in the model of chronic and inflammatory pain. However, the essential oil was devoid of anti-inflammatory activity. Moreover, the essential oil had no analgesic effect in tail flick test as a model of acute pain. The LD50 value of 0.59 ml/kg was obtained for the essential oil. This low toxicity of the essential oil makes it worthy for further studies